Ab1147 Differential Recognition of Topoisomerase 1 (Top1)-Dna Complexes Modulates Activation of Top1-Reactive B Cells in Systemic Sclerosis

Background: Systemic sclerosis (SSc) is hallmarked by autoreactive B cell responses against nuclear antigens. To date, it is poorly understood if and how these B cell responses contribute to disease. A subset of SSc patients harbors B cells targeting the nuclear enzyme topoisomerase 1 (TOP1). Anti-TOP1 autoantibodies (ATAs) associate with severe disease marked by progressive lung and skin fibrosis. Patients with lung fibrosis harbor TOP1-reactive B cells with an activated phenotype 1. Here, we hypothesized that mechanisms underlying the activation of TOP1-reactive B cells could determine the development of disease and drive its progression. Importantly, DNA binding by TOP1 generates TOP1-DNA cleavage complexes (TOP1cc). TOP1cc formation could modulate the antigenicity of TOP1. Objectives: To study the effect of DNA binding by TOP1 (TOP1cc formation) on the recognition by ATAs and the activation of TOP1-reactive B cells. Methods: Monoclonal ATAs (ATA-mAbs, n=25) were generated from patient-derived TOP1-reactive B cells. The reactivity of these ATA-mAbs towards TOP1 and TOP1cc was analyzed by ELISA, western blotting, and mass photometry. Recognition of TOP1 and TOP1cc by polyclonal ATA-IgG/A/M and ATA-IgG subclasses was studied using plasma from ATA-positive SSc patients (n=30), ATA-negative SSc patients (n=6) and healthy donors (n=6). ATA-expressing RAMOS B cell lines were generated to study B cell activation (Syk phosphorylation) upon stimulation with TOP1 and TOP1cc. Results: Individual ATA-mAbs differentially recognized TOP1 and TOP1cc. Most but not all ATA-mAbs (20/25) displayed enhanced recognition of TOP1cc compared to TOP1 in ELISA, a finding which could be replicated by western blotting and mass photometry. Antibodies with enhanced TOP1cc recognition (termed anti-TOP1cc autoantibodies) could be observed in plasma of all ATA-positive SSc patients tested. Anti-TOP1cc autoantibodies were predominantly of the IgG1 and IgM isotypes, but rarely IgA. Anti-TOP1cc-IgG levels correlated with levels of ATA-IgG (rs=0.81, ps=0.67, ps=0.56, p=0.008). Anti-TOP1cc-expressing RAMOS B cells reacted more strongly to antigenic stimulation than anti-TOP1-expressing RAMOS B cells. This effect was dependent on the binding of TOP1 to DNA. Conclusion: The autoreactive B cell response targeting TOP1 in SSc patients demonstrates remarkable heterogeneity that has not been recognized so far. DNA binding enhances recognition of TOP1 by ATA, presumably by changing the conformation of TOP1. This differential recognition of TOP1 and TOP1cc may be clinically relevant as it determines the degree of ATA-expressing B cell activation, a feature linked to SSc disease manifestations and, possibly, disease progression. REFERENCES: 1 Wortel CM, Liem SI, van Leeuwen NM, Boonstra M, Fehres CM, Stöger L, Huizinga TW, Toes RE, De Vries-Bouwstra J, Scherer HU. Anti-topoisomerase, but not anti-centromere B cell responses in systemic sclerosis display active, Ig-secreting cells associated with lung fibrosis. RMD Open. 2023 Jul;9(3):e003148. doi: 10.1136/rmdopen-2023-003148. Acknowledgements: NIL. Disclosure of Interests: None declared.